The proposed investigation considers 4 direct-acting, DNA methylating nitroso compounds. Three of these, methylnitrosourea (MNU), methylnitrosourethane (MNUT) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are known laboratory animal carcinogens. The fourth, nitrosocimetidine (NC), is the nitrosated derivative of "Tagamet", a drug used very widely and successfully in the clinical treatment of gastrointestinal disorders. MNU, MNNG, NC and probably MNUT (to be shown) modify DNA in vitro and in the human cell in culture to produce the same kinds and yield distribution of methylation lesions. The proximal methylating species is believed to be the same in each case. The rates of production of this species, due to compound decomposition, are quite divergent and also show differential thiol sensitivity. These factors may influence biological activity. Increasing decomposition sensitivity to thiol (glutathione) is probably in the order: MNU, MNNG, MNUT, NC. This will be tested using quantitative high pressure liquid chromatography (HPLC) techniques for assessing intact and denitrosated compound. Intracellular thiol involvement in cellular DNA methylation and the dependence of methylation rate on thiol is likely to increase in the same order. This will be tested using human lymphoblastiod cells in culture (Raji cells), HPLC techniques for assessing DNA methylation yields and a biochemical assay for cellular acid-soluble thiol. Thiol dependence will also be checked by modulating cellular glutathione with diethyl mateate. Cellular thiol levels change with growth state thus DNA methylation by the 4 compounds may be differentially dependent on growth state. This will be tested using synchronized human fibroblasts in culture (WI-38 cells), HPLC and biochemical assays. Using Raji cells, inhibition of DNA synthesis (thymidine incorportation) and cell survival (colony-forming efficiency) will be constructed as a function of DNA methylation yield. It is anticipated that these sets of curves will be superimposable. The possibility of a variable reaction specificity at the DNA replication fork due to differential thiol-dependent decomposition will be tested. Also the efficiency of DNA repair in Raji cells as a function of DNA methylation rate will be determined by assessing adduct persistence.